Standard Flow Multiplexed Proteomics (SFloMPro)—An Accessible Alternative to NanoFlow Based Shotgun Proteomics

Multiplexed proteomics using isobaric tagging allows for simultaneously comparing the proteomes of multiple samples. In this technique, digested peptides from each sample are labeled with a chemical tag prior to pooling sample for LC-MS/MS with nanoflow chromatography (NanoLC). The isobaric nature of the tag prevents deconvolution of samples until fragmentation liberates the isotopically labeled reporter ions. To ensure efficient peptide labeling, large concentrations of labeling reagents are included in the reagent kits to allow scientists to use high ratios of chemical label per peptide. The increasing speed and sensitivity of mass spectrometers has reduced the peptide concentration required for analysis, leading to most of the label or labeled sample to be discarded. In conjunction, improvements in the speed of sample loading, reliable pump pressure, and stable gradient construction of analytical flow HPLCs has continued to improve the sample delivery process to the mass spectrometer. In this study we describe a method for performing multiplexed proteomics without the use of NanoLC by using offline fractionation of labeled peptides followed by rapid “standard flow” HPLC gradient LC-MS/MS. Standard Flow Multiplexed Proteomics (SFloMPro) enables high coverage quantitative proteomics of up to 16 mammalian samples in about 24 h. In this study, we compare NanoLC and SFloMPro analysis of fractionated samples. Our results demonstrate that comparable data is obtained by injecting 20 µg of labeled peptides per fraction with SFloMPro, compared to 1 µg per fraction with NanoLC. We conclude that, for experiments where protein concentration is not strictly limited, SFloMPro is a competitive approach to traditional NanoLC workflows with improved up-time, reliability and at a lower relative cost per sample.

Persistence alters the interaction between Chlamydia trachomatis and its host cell.

In response to stress, the obligate intracellular pathogen Chlamydia trachomatis stops dividing and halts its biphasic developmental cycle. The infectious, extracellular form of this bacterium is highly susceptible to killing by the host immune response, and by pausing development Chlamydia can survive in an intracellular, ‘aberrant’ state for extended periods of time. The relevance of these aberrant forms has long been debated, and many questions remain concerning how they contribute to the persistence and pathogenesis of the organism. Using reporter cell lines, fluorescence microscopy, and a di-peptide labeling strategy, we measured the ability of C. trachomatis to synthesize, assemble, and degrade peptidoglycan under various aberrance-inducing conditions. We found that all aberrance-inducing conditions affect chlamydial peptidoglycan, and that some actually halt the biosynthesis pathway early enough to prevent the release of an immunostimulatory peptidoglycan component, muramyl tripeptide. In addition, utilizing immunofluorescence and electron microscopy, we determined that the induction of aberrance can detrimentally affect the development of the microbe’s pathogenic vacuole (the inclusion). Taken together, our data indicate that aberrant forms of Chlamydia generated by different environmental stressors can be sorted into two broad categories based on their ability to continue releasing peptidoglycan-derived, immunostimulatory muropeptides and their ability to secrete effector proteins that are normally expressed at the mid- and late- stages of the microbe’s developmental cycle. Our findings reveal a novel, immuno-evasive feature inherent to a subset of aberrant chlamydial forms and provide clarity and context to the numerous persistence mechanisms employed by these ancient, genetically-reduced microbes.

In vivo monitoring of viable bacteria by SPECT using 99mTc-HYNIC(GH)2-UBI 29-41 and 99mTc-HYNIC(Tricine)2-UBI 29-41

Background: The number of bacterial infections that—for various reasons—are challenging to cure continues to increase. One such reason is persister cell infection. To investigate persister formation and persister infections, viable bacteria must be evaluated in the same animal over time. In this study, the feasibility of monitoring viable bacteria by SPECT using two labeled peptides was evaluated. Results: Two types of ubiquicidin (UBI) 29-41 labeled with technetium-99m, 99mTc-HYNIC(GH)2-UBI 29-41 and 99mTc-HYNIC(Tricine)2-UBI 29-41, were synthesized. The in vitro binding of these labeled peptides to Staphylococcus aureus was measured. For the in vivo study, each labeled peptide was injected into S. aureus infected mouse thigh after treatment with various doses of ciprofloxacin (CPFX). Two hours after injection, the accumulation of each labeled peptide at the infection site was assessed by SPECT, and then the number of viable bacteria was determined from the accumulation detected. The peptide labeling was successful, and the radiochemical purity was 91±9% (GH, n=8) and 100% (Tricine, n=8). The in vitro binding of the labeled peptides to S. aureus (5×108 cfu) without serum was 78.9% (GH) and 85.5% (Tricine) of the total 99mTc activity. With serum, the binding rate was 67.5% (GH) and 13.3% (Tricine). The accumulation of labeled peptide was calculated from the SPECT images, and that in the bacterial infection site (left thigh) was higher than that in the non-infection site (right thigh) for both peptides. Good correlation was found between the target-to-non-target (T/NT) ratios of each labeled peptide and the viable bacterial count at the infection site, and 99mTc-HYNIC(Tricine)2-UBI 29-41 had a wider range than 99mTc-HYNIC(GH)2-UBI 29-41.Conclusion: Using the SPECT/labeled peptide method, it was possible to monitor viable bacterial count in the range 103–108 cfu, which is appropriate for tracking viable bacterial counts in the same animal over time.

Radiolabeled Peptides for SPECT and PET Imaging in the Detection of Breast Cancer: Preclinical and Clinical Perspectives

Breast cancer is the most common cancer in women worldwide. Due to the heterogeneous nature of breast cancer, the optimal treatment and expected response for each patient may not necessarily be universal. Molecular imaging techniques could play an important role in the early detection and targeted therapy evaluation of breast cancer. This review focuses on the development of peptides labeled with SPECT and PET radionuclides for breast cancer imaging. We summarized the current status of radiolabeled peptides for different receptors in breast cancer. The characteristics of radionuclides and major techniques for peptide labeling are also briefly discussed.

Standard Flow Multiplexed Proteomics (SFloMPro). An Accessible and Cost-Effective Alternative to NanoLC Workflows

Multiplexed proteomics using isobaric tagging allows for simultaneously comparing the proteomes of multiple samples. In this technique, digested peptides from each sample are labeled with a chemical tag prior to pooling sample for LC-MS/MS with nanoflow chromatography (NanoLC). The isobaric nature of the tag prevents deconvolution of samples until fragmentation liberates the isotopically labeled reporter ions. To ensure efficient peptide labeling, large concentrations of labeling reagents are included in the reagent kits to allow scientists to use high ratios of chemical label per peptide. The increasing speed and sensitivity of mass spectrometers has reduced the peptide concentration required for analysis, leading to most of the label or labeled sample to be discarded. In conjunction, improvements in the speed of sample loading, reliable pump pressure, and stable gradient construction of analytical flow HPLCs has continued to improve the sample delivery process to the mass spectrometer. In this study we describe a method for performing multiplexed proteomics without the use of NanoLC by using offline fractionation of labeled peptides followed by rapid standard flow HPLC gradient LC-MS/MS. Standard Flow Multiplexed Proteomics (SFloMPro) enables high coverage quantitative proteomics of up to 16 mammalian samples in about 24 hours. In this study, we compare NanoLC and SFloMPro analysis of fractionated samples. Our results demonstrate that comparable data is obtained by injecting 20 micrograms of labeled peptides per fraction with SFloMPro, compared to 1 microgram per fraction with NanoLC. We conclude that, for experiments where protein concentration is not strictly limited, SFloMPro is a competitive approach to traditional NanoLC workflows with improved up-time, reliability and at a lower relative cost per sample.

Comparative Proteome Analysis Reveals Lipid Metabolism-Related Protein Networks in Response to Rump Fat Mobilization

Altay is a typical fat-tailed sheep breed displaying the unique ability to rapidly mobilize fat, which is vital for maintaining a normal metabolism that facilitates its survival in lengthy winter conditions. However, the physiological, biochemical, and molecular mechanisms underlying fat mobilization remain to be elucidated. In this study, the monitoring of rump fat adipocyte sizes disclosed a positive correlation between cell size and fat deposition ability. In addition, we subjected sheep to persistent starvation to imitate the conditions that trigger rump fat mobilization and screened 112 differentially expressed proteins using the isobaric peptide labeling approach. Notably, increased secretion of leptin and adiponectin activated the key fat mobilization signaling pathways under persistent starvation conditions. Furthermore, the upregulation of resistin (RETN), heat-shock protein 72 (HSP72), and complement factor D (CFD) promoted lipolysis, whereas the downregulation of cell death-inducing DFFA-like effector C (CIDEC) inhibited lipid droplet fusion, and the increase in HSP72 and apolipoprotein AI (Apo-AI) levels activated the body’s stress mechanisms. The synergistic actions of the above hormones, genes, and signaling pathways form a molecular network that functions in improving the adaptability of Altay sheep to extreme environments. Our findings provide a reference for elucidating the complex molecular mechanisms underlying rump fat mobilization.